Pioglitazone Inhibits Metal Cluster Transfer of mitoNEET by Stabilizing the Labile Fe-N Bond Revealed at Single-Bond Level.

Outer mitochondrial membrane protein mitoNEET (mNT) is a target of the type 2 diabetes drug pioglitazone. It contains a labile FeS(His)(Cys) metal cluster with a single Fe-N(His87) coordinating bond and can transfer its cluster to acceptor proteins. Previous ensemble studies showed that pioglitazone's binding inhibited the transfer by stabilizing the cluster, and histidine 87 may be the key mediator. Here we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to study the unfolding process of mNT dimer in the absence and presence of pioglitazone, which can distinguish the binding effect for different regions of a protein. By developing a two-step strategy using different mNT monomers with respective purification tags, we solve the problem that the classic polyprotein formation disables the mNT to dimerize. As a result, a polyprotein including a stable, naturally noncovalently bound mNT homodimer is obtained, which is required for reliable AFM measurement and pioglitazone binding. Then, the dissociation rate () of the metal cluster was measured, showing a 10-fold decrease upon pioglitazone binding, while the other parts decreased only 3-fold, verifying that pioglitazone mainly stabilizes the cluster. Moreover, when the Fe(III)-N(His87) bond was ruptured, this effect for the remaining FeS(Cys) intermediate largely disappeared. Consequently, AFM results revealed that pioglitazone inhibited the metal cluster transfer of mNT by stabilizing the labile Fe(III)-N(His87) bond. In addition, an alternative method to build a natural, noncovalently bound protein dimer or complex for reliable single-molecule measurement was developed.